Mycobacteria is a major bacterial pathogen group for man in which are included the etiological agents of tuberculosis and leprosy, respectively Mycobacterium tuberculosis and Mycobacterium leprae. Mycobacterium tuberculosis accounts for more deaths world wide than any other human pathogen. The World Heath Organization estimated that there are approximately 8 million new cases and an annual mortality of 3 million. On the other hand leprosy afflicts 10 to 12 million people in 152 countries. Leprosy is endemic in the Arctic Circle and in Africa, South-America and South-East Asia. M. bovis and M. africanum can also cause tuberculosis in humans. M. bovis causes tuberculosis in ruminants and in other animals and can be transmitted to humans by ingestion of contaminated milk or meat. Tuberculosis associated with M. africanum has been diagnosed in immigrants from Central African countries.
Many species of mycobacteria other than M.tuberculosis, M. africanum, M. bovis and M. leprae present in the environment can also cause human diseases. For example, pulmonary diseases may be caused by the M. kansasii, M. xenopi and M.avium complex. M. marinum, often isolated from aquaria and swimming pools, may cause chronic granulomas of the skin.
On the other hand BCG, an avirulent strain of M. bovis, is widely used as a vaccine against tuberculosis. This bacillus was developed by Calmette and Guxc3xa9rin who noted a change in colony morphology after a serial passage of a virulent strain of M. bovis in a potato glycerin medium containing ox bile. The efficacy of BCG continues to be debated. BCG protects against serious and disseminated disease including meningitis in newborn babies. However, its efficiency to prevent pulmonary forms, particularly in adults, was shown to be variable.
The main advantage of live vaccines is that they can persist in the host owing to their capacity to replicate, resulting in continuous exposure of the host to the vaccine antigen. Thus, it is possible that a single dose of live vaccine could represent an effective dose for the induction of a long-lasting immune response. A vast array of bacterial species have been attenuated by a variety of means in an attempt to develop both safe and effective vaccines and carrier strains for heterologous vaccine antigens. The BCG is the only bacterial live vaccine that has been extensively used in humans. In the past 40 years it has been administered to more than 2.5 billion people with very few adverse effects. BCG offers some other unique attributes for the development of live vaccine vehicles: 1. it can be given at birth or shortly after; 2. it is able to induce a long-lasting immune response; 3. it is a potent immunological adjuvant; 4. BCG can be administrated as an oral vaccine to stimulate mucosal immunity; 5. it is heat stable; BCG does not require conservation at low temperatures; 6. it is easy and inexpensive to produce.
The development of genetic tools for mycobacteria has enabled the cloning and the expression of foreign genes in fast-growing M. smegmatis mycobacteria and in M. bovis BCG. Plasmids have been identified in several species of mycobacteria such as M. avium, M. scrofulaceum and M. fortuitum, though the functions of most of them are unknown. The most thoroughly studied mycobacterial plasmid was the cryptic plasmid pAL5000 isolated from a strain of M.fortuitum by Labidi and co-workers 1984. The analysis of the complete sequence of the pAL5000 revealed 5 ORF and an origin of replication (Rauzier, J., Moniz-Pereira, J., and Gicquel, B. Gene 71:315-321, (1988)). This information was essential for the construction of small shuttle vectors for E.coli mycobacteria, which include an origin of replication functional in E. coli (OriE) derived from pUC19. As a genetic marker the Tn903-derived aph gene was used, which confers kanamycin resistance to the transformants. Expression cassettes derived from the promoter regions of BCG hsp-60 (Thole, et al., Infect. and Immun., Vol.55, p.1466-1475 (1987)) and hsp-70 (Young, R.) genes ( M.tuberculosis 19-kDa antigen (Ashbridge, K. R et al., Nucleic Acids Res. Vol. 17: 1249.1989. Stover, K. et al., J. Exp. Med., Vol 178: 197-209. (1993)), M. fortuitum bla gene (Timm, J. et al., Mol.Microbiol. Vol. 12: 491-504. (1994)) among others, were successfully used for the expression of foreign genes in fast-growing mycobacteria and in BCG.
The use of recombinant mycobacteria as multivalent vaccine vehicles requires a stable construction for a long expression of the antigen genes following the administration of the bacteria to the host.
Antigenic-expressing plasmid vectors are not very stable in the absence of selection, although they reach high levels of expression of the foreign DNA.
One of the possibilities for increasing the stability of recombinant bacterial strains is to use a site-specific integration system of temperate bacteriophages. By this process a heterologous DNA covalently linked to a phage DNA integration locus can be inserted into the bacterial genome and be maintained for a long time.
The temperate mycobacteriophage Ms6, isolated from the strain of Mycobacterium smegmatis HB5688, is able to infect and to lysogenize some strains of M. smegmatis. It was shown that Ms6 can be used as a vehicle for the insertion of foreign DNA into mycobacteria genome. The aminoglycoside phosphotransferase gene from the Tn5 transposal was inserted into the M. smegmatis genome through lysogenization using a transducing derivative of the mycobacteriophage Ms6. It was demonstrated that Ms6 DNA can be inserted into the bacterial genome through a site-specific recombination mechanism. The DNA restriction fragment that contains the phage attachment site attP was identified and sequence analysis of this region showed that Ms6 is different from D29 and L5 mycobacteriophages. The integrative locus of mycobacteriophage Ms6 was sequenced and inserted into a plasmid vector. This recombinant plasmid was integrated into Mycobacterium smegmatis, M. bovis-BCG, M tuberculosis. The present invention was based on the site-specific integration system of the mycobacteriophage Ms6.
The object of the present invention is the construction of highly stable recombinant mycobacteria strains, particularly BCG, to be used as xe2x80x9cin vivoxe2x80x9d vectors for presenting antigens, immunomodulators and other therapeutic agents.
The present invention relates to: 1. the Ms6 DNA integrating region responsible for the integration of the bacteriophage DNA into the mycobacterial genome; 2. a second DNA fragment that includes the Ms6 attP region linked to an expression cassette composed of the relevant DNA sequence (which may encode a variety of heterologous genes, including genes for antigens which protect against several pathogens, lymphocynes and other therapeutic agents) under the control of a promoter region; 3. a third DNA fragment that carries the integrase gene under the control of a strong promoter region originating from the bacteriophage Ms6; 4. a new method of integrating DNA based on the transient production of integrase sufficient for directing the integration of the relevant DNA sequence linked to the attP region into the bacterial genome; 5. the expression of vaccine-stable vehicles which are such recombinant strains of mycobacteria produced by this new integrating process capable of continuously expressing the relevant DNA fragment.
The Ms6 DNA integrating region (attP-int) includes: the phage attachment site (attP) with the bacterial genome and the gene of the integrase, which encodes the enzyme that mediates the recombination between the attP and a corresponding attB site of the bacterial genome. This mechanism is called site-specific integration because it only occurs in a particular site of the bacterial genome. A common core sequence is present in both the attP and attB regions and is the site where the DNA exchange occurs. This recombinational event leads to the duplication of the common core sequence which will flank the inserted DNA.
The DNA integration process of the subject invention has important advantages over the integrating systems that are presently available. In particular, an improvement of the transformation efficiency of mycobacteria due to the use of a bacteriophage integrase in the integration process. This differs from the process that is presently available in which the DNA fragment is integrated into the bacterial genome by a double homologous recombination mechanism which, in slow growing bacteria such as BCG, occurs with a very low frequency, resulting in poor transformation efficiency.
An integrative vector based on a mycobacteriophage attP-int region linked to an expression cassette is available to construct recombinant mycobacteria strains. However, considering that in bacterial lysogens the excision process of the prophage requires the contribution of the integrase and the excisionase, it is possible to improve the stability of the recombinant strains by eliminating the integrase gene from the integrative vector. This is the subject of the present invention. A new process is proposed which eliminates the integrase gene from the integrative vector and therefore from the genome of the recombinant mycobacteria produced to guarantee the stability of the recombinant strains. This novel integration process consists in transforming the mycobacterial cells simultaneously with two plasmids. Plasmid 1 includes the integrase gene under the control of a strong promoter region and it is not capable of replication because it does not have a mycobacterial origin of replication. Plasmid 2 contains the attP region linked to a relevant DNA which is under the control of mycobacterial gene regulation signals. The supply of the integrase gene in an independent suicide vector produces enough integrase to direct the integration of plasmid 2 which carries the attP region and the relevant DNA.
It was confirmed that this process of integrating relevant DNA into the bacterial genome produces recombinant mycobacterial strains that do not lose the exogenous DNA following long non-selective growth. Therefore, these highly stable recombinant mycobacterial strains can be safely used as vehicles to express, following administration to humans or animals, a gene or genes of interest. These recombinant mycobacterial strains can be used as vectors for presenting one or more protective antigens, against one or more pathogenic agents. These mycobacterial vehicles can also be used to express immunopotentiators, cytosines, anti-tumour and anti-cancerous agents and other useful polypeptides and proteins.
The integrative vectors and the integrating process described in this invention have made it possible to insert exogenous DNA into the genome of a wide range of Mycobacterium species, including: M.smegmatis, M.vaccae, M. tuberculosis, M. bovis-BCG.The scope of the present invention is not restricted to mycobacteria and is applicable to other bacteria, such as: Salmonella ssp., Vibrio spp., Shigella spp., Lactobacillus spp., Streptomyces spp., Corynebacterium spp., Listeria spp.